Fig. 2

Heparan sulfate regulates flow-mediated RFPEC eNOS-pS1177 expression and colocalization with cav-1. En face z-projections are shown for red fluorescent cav-1 (a, d, g, j, m), green fluorescent eNOS-pS1177 (b, e, h, k, n), and merged cav-1 with eNOS-pS1177 (c, f, i, l, o). Conditions examined include static flow (a–c), DF (d–f), UF (g–i), static flow with Hep III treatment (j–l), and UF with Hep III treatment (m–o). Arrows are pointing to areas of significant cav-1 colocalization with eNOS-pS1177. Scale bar = 30 μm. p Quantification of eNOS-pS1177 expression. Compared to static controls, cells with intact HS display decreased eNOS-pS1177 expression when exposed to DF, but increased eNOS-pS1177 expression when exposed to UF. Upon enzymatic degradation of HS, exposure to UF results in no significant increase in eNOS-pS1177 expression, compared with the effects of static flow and DF. q Quantification of colocalization shows significant differences in eNOS-pS1177 overlap with cav-1 when RFPECs in UF conditions are compared to RFPECs in DF and static flow conditions. RFPECs exposed to static and DF conditions along with enzymatically degraded HS show lower cav-1 and eNOS-pS1177 colocalization that is statistically different to RFPECs with intact HS in UF conditions. p, q Number of independent experiments “N” are shown for each condition, and *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 are indicated